I have experienced that swim up technique for sperm separation during IVF in ovine was not suitable for ovine sperm? So I had to use percoll gradient (90 and 45%) for ovine sperm separation, although I used to use swim-up separation in bovine IVF. And I obtained good results with percoll gradient compared with swim up technique in ovine IVF. I want to know whether or not you have ever faced similar situations during IVF? Also what kind of separation technique do you prefer to separate semen in species (especially goat, sheep and cattle) in IVF? Have you ever used one gradient (i.e. only %45) percoll technique?
Thank you Navid for sharing your experience,
It is interesting we have similar experiences in ram semen. Do you think the reason of this? Ram spermatozoa may be more vulnerable to waiting periods in swim up.
I agree with you about only 45% percoll separation technique. I have not tried this method but some researchers use this method and they say to have obtained good results. However, in my opinion dead or defected spermatozoa go down to tube with good spermatozoa.
My best wishes,
Umut
Dear Omer Ucar,
Thank you for your knowledge sharing about percoll usage.
Normally, I dilute percoll with 10x TALP solution, 9:1 ratio. Then I obtain 90%Percoll and if I want to use 45% Pecoll, I dilute again 90% Percoll with normal (iso-tonic) Talp 1:1 ratio.
It is interesting way to obtain 45% Pecoll via dilution of (isotonic) TALP with 100%Pecoll. I think osmotic pressure of Percoll may be lower than iso-osmotic presure of semen. It may be detrimental to spermatozoa via hipo-ostmotic pressure of percoll solution. Do you face any problem when you use 45% percoll in your way?
My best wishes,
Umut
NO obvious problem we have observed at all. It was also OK with BOVINE semen too. It means that, with %45 Percoll (in TALP), there appears to be no clear species difference (for canine, ovine and bovine) of the resultant pellet (containing mostly the live sperm cells) in the preference of centrifugation medium. You can check the actual osmotic pressures of both percoll and TALP or its mixture (45% P. plus 55% T.) and the cellular characteristics of the pellet (viability, morphology or functionality, thermo-resistance, etc) as you may wish. But, I would rather suggest you to choose a *working protocol* for semen processing. If you try to develop the best centrifugation solution (with optimum conditions nearest possible) go for further lab-based trials on the bench. Regards.. Omer
Dear Omer,
I will try your mixture of 45% Percoll with 55% TALP solution as soon as possible. And I will contact with you again about what have been happened.
I agree with you we have to try every biological process before real study experiment. An other words we have to carry out preliminary study before real work. In my experience there had been no any problem with swim-up procedure until I tried swim-up for ram semen in IVF. I faced problem with swim-up and I had to change my sperm separation protocol to Percoll Gradient about which I had not tried and I had no experiment before. I obtained good results with Pecoll in ovine IVF. Maybe, Percoll could have been some toxic effects on ram semen and then I need have used other sperm separation techniques i.e. glass wool, maybe swim down etc.
Warm regards,
Umut
Hi Umut,
the density gradient centrifugation and the swim-up are very good methods but they have one limitation:they can not identify and eliminate apoptotic sperm cells. Many stuides have shown that apoptotic sperm potentially decrease the chance of success for IVF and pregnancy.
The MACS ®ART Annexin V System is easy to perform, fast and removes apoptotic sperm effectively. You should perform DGC prior to the kit.
Kind regards
Sandra
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